Notch receptors initiate a signaling pathway essential for development in all metazoans. Defects in the pathway cause a number of congenital birth defects and cancers. Notch can function as either an oncogene or tumor suppressor in cancers, so therapies that can increase or decrease Notch activity are needed. Notch is regulated at numerous levels, but glycosylation of the Notch extracellular domain (ECD) has emerged as a major regulator that can increase or decrease Notch activity depending on context. The Notch ECD contains up to 36 tandem Epidermal Growth Factor-like (EGF) repeats, many of which contain consensus sequences for O-linked fucose. O-Fucose is added to EGF repeats by Protein O-fucosyltransferase 1 (POFUT1) and is essential for Notch function in all contexts examined, while extension of O-fucose by the Fringe family of ?3-N- acetylglucosaminyltransferases is modulatory. Fringe regulation of Notch has become a paradigm for regulation of a signaling receptor by altering its glycosylation status. Fringe modifications typically enhance Notch1 (N1) activation by the Delta-like family of ligands (DLL1, DLL4), but inhibit activation by Jagged ligands (J1, J2). In the past grant cycle we made significant progress on the molecular mechanisms by which Fringe modifications differentiate between ligands by identifying which EGF repeats of N1 are modified by each Fringe enzyme, and determining which of those EGF repeats are responsible for the modulatory effect. Our results demonstrate that Fringes ?mark? the Notch ECD, with some marks activating DLL1-N1 activation, and distinct marks inhibiting J1-N1 activation. In addition, we have recently shown that fucose analogs (fucose with modifications to carbon 6) inhibit Notch activation in a ligand-specific manner, providing proof of concept that we can use small molecules to alter Notch glycosylation and regulate its function. Our results have led to the overall hypothesis for this application: O-glycans at specific sites on Notch regulate its activity by directly modulating initial Notch-ligand binding, or by modulating events subsequent to ligand binding but prior to proteolytic receptor activation. We will test this hypothesis in three aims. Aim 1 seeks to address how Fringe modifications inhibit J1-N1 activation. Our published work shows that Fringes enhance binding of DLL1 to N1, providing a molecular explanation for enhanced DLL1-N1 activation, but we also showed that Fringes enhance J1 binding to N1. Thus, Fringe modification must inhibit J1-N1 activation by affecting a step subsequent to ligand binding but prior to proteolytic receptor activation. The experiments in Aim 1 will examine several possible mechanisms for this effect. Aim 2 examines the basis for the striking site-specific elongation of O- fucose on Notch EGF repeats by the Fringes. This knowledge will allow us to eliminate Fringe modification at specific sites without affecting addition of O-fucose. Finally, Aim 3 examines how the fucose analogs affect Notch activity, whether we can generate more potent analogs with distinct activities, and whether we can use the analogs to inhibit growth of cancer cells dependent on Notch activity for division.